Compositions and methods for treating ophthalmic disorders

ABSTRACT

The present invention relates to compositions and methods for treating ophthalmic disorders of humans or animals. The present compositions and methods are highly suitable for intraocular administration into the interior of an eye and provide therapeutic effects to the eye as they are effective in stabilizing, enhancing and/or improving a patient&#39;s vision. More specifically, the present invention relates to compositions and methods for treating ophthalmic diseases or disorders with exudative, hemorrhagic and/or inflammatory conditions. Even more specifically, the present invention relates to compositions and methods for treating retinal diseases or disorders, and more specifically ophthalmic diseases or disorders related to impaired retinal vessel permeability and/or integrity.

The present invention relates to compositions and methods for treatingophthalmic disorders of humans or animals. The present compositions andmethods are highly suitable for intra- and peri-ocular administrationinto the interior of an eye and provide therapeutic effects to the eyeas they are effective in stabilizing, enhancing and/or improving apatient's vision. More specifically, the present invention relates tocompositions and methods for treating ophthalmic diseases or disorderswith an exudative and/or inflammatory condition. Even more specifically,the present invention relates to compositions and methods for treatingretinal diseases or disorders, and more specifically ophthalmic diseasesor disorders related to impaired retinal vessel permeability and/orintegrity.

Ophthalmic diseases or disorders in general terms can be divided into(i) front-of-eye diseases or disorders such as, for example, cornealoedema, anterior uveitis, pterygium, corneal diseases or opacificationswith an exudative or inflammatory component, conjunctivitis, allergy andlaser induced exudation and (ii) back-of-eye diseases or disorders suchas, for example, exudative eye diseases and more particularly exudativeretinopathies, exudative macular degeneration, macular oedema, diabeticretinopathy, age-related macular degeneration or retinopathy ofprematurity.

The pathogenesis of exudative and/or inflammatory eye diseases ordisorders, and more particularly of exudative retinopathies, involvesblood-retinal barrier (BRB) alteration and inflammation. The retinaessentially consists in neuronal matter, and the barrier between theretina and the choroidal vascular system, herein referred as BRB isquite similar to the blood-brain barrier. The BRB is made up of twocompartments defined as follows: an inner barrier consisting of retinalvascular endothelial cells that line the blood vessels of the choroidand an outer barrier consisting of the retinal pigment epithelial (RPE)cells that separate the choroid from the retina. Functionally, the BRBis dependent on the integrity of the RPE, the retinal vasculature andassociated glial cell layers which behave as an additional componentpreventing the direct access of blood vessels to the neuronalenvironment. The BRB functions to preserve the physiological environmentof the neuronal retina. When the BRB is compromised, plasma leaks acrossthe BRB into the retina thus contributing to pathological processes suchas exudative retinopathies and vision impairment. Ailments associatedwith breakdown of the BRB in the posterior region of the retina include,for example, oedematous retinal conditions such as, myopicretinopathies, macular oedema such as clinical macular oedema orangiographic cystoid macular oedema arising from various aetiologiessuch as diabetes, exudative macular degeneration and macular oedemaarising for example from laser treatment of the retina.

Other conditions can lead to or be associated with exudativeretinopathy. For example, myopic retinopathy is a condition that resultsfrom severe malformation of the retina in part due to overgrowth of thesclera. This deformation leads to restriction of the blood vesselsnetwork within the choroid, and ultimately to a process of compensatoryneovascularisation. Nevertheless, the newly formed vessels appearfragile and prone to leakage and exudation, leading to exudativeretinopathy.

Similarly, macular oedema (e.g. clinical macular oedema or angiographiccystoid macular oedema) is a condition involving swelling of the maculaand typically occurs as a result of aetiologies such as disease (e.g.diabetes), injury or eye surgery. Fluid collects within the layers ofthe macula, causing blurred, distorted central vision.

In exudative macular degeneration (also known as “wet” or neovascularage-related macular degeneration (wet-AMD)) abnormal overgrowth of bloodvessels from the choroid into the retina occurs, compromising the BRB.The abnormal blood vessels are fragile and prone to leakage.

Diabetic retinopathy is a severe complication of diabetes. In theinitial stage, capillary microaneurysm and dot haemorrhage are observed.Thereafter, microvascular obstruction and retinal oedema result fromvascular hyperpermeability and neovascularization. In the last stage,retinal detachment is caused by the traction of connective tissues grownin the vitreous body. Further, iris rubeosis and neovascular glaucomaare observed, leading to blindness.

Retinal ischemia or degeneration is another retinopathy. It may result,for example, from injury, trauma, tumours or be associated with variousdisorders such as occlusion of a blood vessel or elevated intraocularpressure which reduces availability of blood, oxygen or other nutrientsto the retina or optic nerve thus leading to neuronal cell death(degeneration) and loss of vision. Such disorders include e.g. diabetes,atherosclerosis, venous capillary insufficiency, obstructive arterialand venous retinopathies (e.g. Retinal Venous Occlusion), glaucoma andsenile macular degeneration.

Treatment of such diseases currently focuses on removing or inhibitingvascular growth by laser treatment, drug therapy or a combination ofboth.

Currently, the most widely used treatment for these disorders is lasertherapy which is directed to removal, destruction or blockage of bloodvessels via photodynamic therapy or laser photocoagulation. For example,focal laser treatment may be applied to micro-aneurysms identified indiabetic retinopathy. Laser therapy is believed to inhibitneovascularisation and to decrease the extent of oedema. However, acomplication of laser treatment is inflammation that may lead to furtheroedema and destruction of large portion of retina with significant riskof vision alteration. In addition, laser treatment is not always apermanent cure as blood vessels may grow again, and micro-aneurysms mayreoccur. Furthermore, laser treatment of abnormal blood vessels cannotbe performed on vessels located in certain retinal areas, such as thecentral region of the macula.

Drug compounds for treating these ophthalmic disorders have beenproposed which have anti-angiogenic or angiostatic properties, such ascorticosteroid (e.g. anecortave acetate, triamcinolone, . . . ).However, corticosteroids have serious side effects that limit their use,for example increase of intra occular pressure (glaucoma) and cataractformation. Other products are directed against vascular endothelialgrowth factor (VEGF) such as Lucentis™ also named ranibizumab orMacugen™ also named pegaptanib sodium. However, to date there isinsufficient evidence to indicate how successful these compounds willbe.

The present invention intends to provide improved compounds and methodsfor the treatment of ophthalmic disorders that at least slow the rate ofdevelopment of said ophthalmic disorders and address the principalproblem underlying these diseases (i.e. retinal vascular permeabilityand/or exudation of fluids from vessels and retinal microvessel ruptureleading to focal hemorrhages). In one aspect of the present invention,are provided compounds and methods for treating ophthalmic disorders,and more specifically exudative and/or inflammatory ophthalmicdisorders. In a more specific aspect of the present invention, areprovided compounds and methods for treating back of the eye diseasesand/or disorders, and more specifically retinal diseases, and even morespecifically ophthalmic disorders related to impaired retinal vesselpermeability and/or stability.

In animals, proteases (e.g. kallikrein, plasmin, elastase, urokinaseplasminogen activator, thrombin, human lipoprotein-associatedcoagulation inhibitor or coagulation factors) are involved in a broadrange of biological pathways affecting blood flow and are thus essentialin wound healing, extracellular matrix destruction, tissuereorganization, and in cascades leading to blood coagulation,fibrinolysis, and complement activation. Proteases are released byinflammatory cells for destruction of pathogens or foreign agents, andby normal and cancerous cells as they move through their surroundings.Overproduction or lack of regulation of proteases activity can havedeleterious consequences leading to pathological conditions. Forexample, kallikreins are serine proteases found in both tissues andplasma, and it has been shown that plasma kallikrein is involved incontact-activated coagulation, fibrinolysis, hypotension, andinflammation (See Bhoola, et al., 1992, Pharmacological Reviews, 44,1-80).

The activity of proteases is regulated by inhibitors. It has been shownthat 10% of the proteins in blood serum are protease inhibitors (Robertset al., 1995, Critical Reviews in Eukaryotic Gene Expression, 5,385-436). Inhibitors of proteases, and more particularly of specificserine proteases, therefore have received attention as potential drugtargets for various pathological situations, such as ischemic diseases,bleeding episodes (e.g. fibrinolysis or fibrinogenolysis, excessivebleeding associated with thrombolytics, post-operative bleeding andinappropriate androgenesis). One such inhibitor for example, aprotinin(also called bovine pancreatic trypsin inhibitor) has been approved inthe United States for prophylactic use in reducing perioperative bloodloss and the need for transfusion in patients during coronary arterybypass graft (for a review see Engles, 2005, Am J Health Syst Pharm.,62, S9-14). The effectiveness of aprotinin is actually associated withits relatively non-specific abilities to inhibit a variety of serineproteases, including plasma kallikrein and plasmin. Kallikrein, a serineprotease, is an enzyme that initiates the CAS cascade leading toactivation of neutrophils, plasmin, coagulation, and various kinins. Itis secreted as a zymogen (pre-kallikrein) that circulates as an inactivemolecule until activated by a proteolytic event early in the contactactivation cascade.

Protease inhibitors are classified into a series of families based onextensive sequence homologies among the family members and theconservation of intrachain disulfide bridges (for review, see Laskowskiand Kato, 1980, Ann. Rev. Biochem. 49, 593-626). Serine proteaseinhibitors of the Kunitz family (i.e. Kunitz type serine proteaseinhibitors) are characterized by their homology with aprotinin (bovinepancreatic trypsin inhibitor). The Kunitz type serine proteaseinhibitors, includes inhibitors of trypsin, chymotrypsin, elastase,kallikrein, plasmin, coagulation factors XIa and IXa, and cathepsin G.These inhibitors thus regulate a variety of physiological processes,including blood coagulation, fibrinolysis, complement activation,inflammation and tumor development. The Kunitz type serine proteaseinhibitors are generally basic, low molecular weight proteins comprisingone or more, native or non native, inhibitory domains (“Kunitzdomains”). The Kunitz domain is a folding domain of approximately 50-60residues, which forms a central anti-parallel beta sheet and a shortC-terminal helix (see e.g. U.S. Pat. No. 6,087,473). This characteristicdomain comprises six cysteine residues that form three disulfide bonds,resulting in a double-loop structure. Between the N-terminal region andthe first beta strand resides the active inhibitory binding loop. Thisbinding loop is disulfide bonded through a Cys residue to the hairpinloop formed between the last two beta strands. Isolated Kunitz domainsfrom a variety of proteinase inhibitors display an inhibitory activity(e.g., Petersen et al., 1996, Eur. J. Biochem. 125, 310-316; Wagner etal., 1992, Biochem. Biophys. Res. Comm. 186, 1138-1145). Linked Kunitzdomains also have an inhibitory activity (see for example U.S. Pat. No.6,087,473). Proteinase inhibitors comprising one or more Kunitz domainsinclude tissue factor pathway inhibitor (TFPI), tissue factor pathwayinhibitor 2 (TFPI-2), amyloid β-protein precursor (AβPP), aprotinin, andplacental bikunin.

The present invention is based on the discovery that inhibitors ofserine proteases, such as, for example, kallikrein, can successfully beemployed to treat ophthalmic disorders, and more specifically exudativeand/or inflammatory ophthalmic disorders. According to one specialembodiment, said inhibitors are peptides that inhibit serine proteases,such as, for example, kallikrein. Similarly, it has been shown that saidinhibitors (e.g. said peptides) can successfully be employed to treatback of the eye diseases, and more specifically diseases related toimpaired retinal vessel permeability and/or integrity (e.g. retinaldegeneration). More specifically, the invention provides methods ofusing kallikrein inhibitors in a method for treating and/or preventingophthalmic disorders and compositions for such use. The invention alsorelates to methods for reducing, inhibiting or preventing exudativeand/or inflammatory conditions in the eye, and more particularly in theback of the eye and compositions for such use.

According to a first embodiment, the Invention provides an ophthalmiccomposition useful for intraocular placement in an eye of a patientcomprising a therapeutically effective amount of at least one peptidethat inhibits serine protease and an ophthalmically compatible solventcomponent.

According to another embodiment, said ophthalmic composition furthercomprises a biocompatible polymeric component in an amount effective todelay release of the said peptide into the interior of the eye after thecomposition is intraocularly placed in the eye; and an ophthalmicallycompatible solvent component in an amount effective to solubilize thepolymeric component, the composition being effective, after beingintraocularly placed into the interior of the eye, to form a delayedrelease composition effective to delay the release of the said peptidein the eye relative to intraocular placement of a substantiallyidentical composition without the polymeric component.

According to another embodiment, the present invention relates to amethod for the prophylactic or therapeutic treatment of ophthalmicdisorders in a patient in need of such treatment that comprises the stepof administering a composition comprising a therapeutically effectiveamount of at least one peptide that inhibits serine protease in saidpatient.

According to another embodiment, the present invention relates to amethod for reducing, inhibiting or preventing exudative and/orinflammatory conditions in the eye, and more particularly in the back ofthe eye and compositions for such use, wherein said method comprises thestep of administering a composition comprising a therapeuticallyeffective amount of at least one peptide that inhibits serine proteasein a patient in need thereof.

According to another embodiment, the present invention relates to theuse of at least one peptide that inhibits serine protease for thepreparation of an ophthalmic composition useful for the prophylactic ortherapeutic treatment of ophthalmic disorders in a patient, and morespecifically those cited above.

According to one specific embodiment, said serine protease in all theabove is kallikrein.

According to another specific embodiment, said serine protease in allthe above is plasma kallikrein.

According to another specific embodiment, said peptides of the Inventionthat inhibits serine protease are kallikrein inhibitors, more preferablyKunitz domain polypeptides.

According to one specific embodiment, said peptide of the Invention thatinhibits serine protease includes (or consists of) the amino acidsequence:

Xaa1 Xaa2 Xaa3 Xaa4 Cys Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Gly Xaa13 CysXaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26Xaa27 Xaa28 Xaa29 Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly Gly Cys Xaa39Xaa40 Xaa41 Xaa42 Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48 Xaa49 Xaa50 CysXaa52 Xaa53 Xaa54 Cys Xaa56 Xaa57 Xaa58 (SEQ ID NO:1), or a fragment orvariant thereof, e.g. a fragment that binds and inhibits kallikrein. Forexample, the peptide can have fewer than 80, 70, 65, 60, 58, 55 or 52amino acids.

“Xaa_(s)” refers to positions in a peptide sequence and are,independently from one another, any amino acid.

According to a specific embodiment, Xaa can by any amino acid exceptcysteine.

According to other specific embodiments, one or more of the followingapply:

Xaa1, Xaa2, Xaa3, Xaa4, Xaa56, Xaa57 or Xaa58 are, independently fromone another, any amino acid or absent;

Xaa10 is an amino acid selected from the group consisting of Asp andGlu;

Xaa11 is an amino acid selected from the group consisting of Asp, Gly,Ser, Val, Asn, Ile, Ala and Thr;

Xaa13 is an amino acid selected from the group consisting of Arg, H is,Pro, Asn, Ser, Thr, Ala, Gly, Lys and Gln;

Xaa15 is an amino acid selected from the group consisting of Arg, Lys,Ala, Ser, Gly, Met, Asn and Gln;

Xaa16 is an amino acid selected from the group consisting of Ala, Gly,Ser, Asp and Asn;

Xaa17 is an amino acid selected from the group consisting of Ala, Asn,Ser, Ile, Gly, Val, Gln and Thr;

Xaa18 is an amino acid selected from the group consisting of His, Leu,Gln and Ala;

Xaa19 is an amino acid selected from the group consisting of Pro, Gln,Leu, Asn and Ile;

Xaa21 is an amino acid selected from the group consisting of Trp, Phe,Tyr, His and Ile;

Xaa22 is an amino acid selected from the group consisting of Tyr andPhe;

Xaa23 is an amino acid selected from the group consisting of Tyr andPhe;

Xaa31 is an amino acid selected from the group consisting of Glu, Asp,Gln, Asn, Ser, Ala, Val, Leu, Ile and Thr;

Xaa32 is an amino acid selected from the group consisting of Glu, Gln,Asp Asn, Pro, Thr, Leu, Ser, Ala, Gly and Val;

Xaa34 is an amino acid selected from the group consisting of Thr, Ile,Ser, Val, Ala, Asn, Gly and Leu;

Xaa35 is an amino acid selected from the group consisting of Tyr, Trpand Phe;

Xaa39 is an amino acid selected from the group consisting of Glu, Gly,Ala, Ser and Asp;

Xaa40 is an amino acid selected from the group consisting of Gly andAla;

Xaa43 is an amino acid selected from the group consisting of Asn andGly;

Xaa45 is an amino acid selected from the group consisting of Phe andTyr;

Xaa6, Xaa7, Xaa8, Xaa9, Xaa20, Xaa24, Xaa25, Xaa26, Xaa27, Xaa28, Xaa29,Xaa41, Xaa42, Xaa44, Xaa46, Xaa47, Xaa48, Xaa49, Xaa50, Xaa52, Xaa53 andXaa54 are, independently from one another, any amino acid.

According to another specific embodiment, each of the first and/or lastfour amino acids of SEQ ID NO:1 can optionally be present or absent andcan be any amino acid, if present, e.g., any non-cysteine amino acid.

According to another specific embodiment, each of the first and/or lastthree amino acids of SEQ ID NO:1 can optionally be present or absent andcan be any amino acid, if present, e.g., any non-cysteine amino acid.

According to another specific embodiment, it is possible to remove one,two, three, or four amino acids from the N-terminus of an amino acidsequence described herein, and/or one, two, three, four, or five aminoacids from the C-terminus of an amino acid sequence described herein.

According to another specific embodiment, the peptide of the Inventionhas a sequence with one or more of the following properties: Xaa11 is anamino acid selected from the group consisting of Asp, Gly, Ser or Val;Xaa13 is an amino acid selected from the group consisting of Pro, Arg,His or Asn; Xaa15 is an amino acid selected from the group consisting ofArg or Lys; Xaa16 is an amino acid selected from the group consisting ofAla or Gly; Xaa17 is an amino acid selected from the group consisting ofAla, Asn, Ser or Ile; Xaa18 is an amino acid selected from the groupconsisting of His, Leu or Gln; Xaa19 can be Pro, Gln or Leu; Xaa21 is anamino acid selected from the group consisting of Trp or Phe; Xaa31 isGlu; Xaa32 is an amino acid selected from the group consisting of Glu orGln; Xaa34 is an amino acid selected from the group consisting of Ile,Thr or Ser; Xaa35 is Tyr; and Xaa39 is an amino acid selected from thegroup consisting of Glu, Gly or Ala.

According to another specific embodiment, the peptide of the Inventionincludes the following amino acids: Xaa10 is Asp; Xaa11 is Asp; Xaa13 isan amino acid selected from the group consisting of Pro or Arg; Xaa15 isArg; Xaa16 is an amino acid selected from the group consisting of Ala orGly; Xaa17 is Ala; Xaa18 is His; Xaa19 is Pro; Xaa21 is Trp; Xaa31 isGlu; Xaa32 is Glu; Xaa34 is an amino acid selected from the groupconsisting of Ile or Ser; Xaa35 is Tyr; and Xaa39 is Gly.

According to the present Invention, it is possible to use all or part ofthe peptides described herein. For example, peptides of the Inventioncan include binding domains for specific kallikrein epitopes. Forexample, the binding loops of Kunitz domains can be cyclized and used inisolation or can be grafted onto another domain, e.g., a framework ofanother Kunitz domain.

Examples of peptides according to the present Invention are described bythe following (where not indicated, “Xaa” refers to any amino acid, anynon-cysteine amino acid or any amino acid from the same set of aminoacids that are allowed for SEQ ID NO:1): (SEQ ID NO:2) Met His Ser PheCys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp PhePhe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu GlyAsn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp,(SEQ ID NO:3) Met His Ser Phe Cys Ala Phe Lys Ala Xaa10 Xaa11 Gly Xaa13Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Arg Xaa21 Phe Phe Asn Ile Phe Thr ArgGln Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly Gly Cys Xaa39 Gly Asn Gln AsnArg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ IDNO:4) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys AlaAsn His Leu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe SerTyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp, (SEQ ID NO:5) Met His Ser Phe Cys Ala Phe LysAla Asp Asp Gly His Cys Lys Ala Asn His Gln Arg Phe Phe Phe Asn Ile PheThr Arg Gln Cys Glu Glu Phe Thr Tyr Gly Gly Cys Gly Gly Asn Gln Asn ArgPhe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:6)Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala Asn HisGln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln Phe Thr Tyr GlyGly Cys Ala Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys MetCys Thr Arg Asp, (SEQ ID NO:7) Met His Ser Phe Cys Ala Phe Lys Ala AspAsp Gly His Cys Lys Ala Ser Leu Pro Arg Phe Phe Phe Asn Ile Phe Thr ArgGln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe GluSer Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:8) Met HisSer Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala Asn His Gln ArgPhe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly CysGly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys ThrArg Asp, (SEQ ID NO:9) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp GlyHis Cys Lys Gly Ala His Leu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser LeuGlu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:10) Met His Ser PheCys Ala Phe Lys Ala Asp Asp Gly Arg Cys Lys Gly Ala His Leu Arg Phe PhePhe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu GlyAsn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp,(SEQ ID NO:11) Met His Ser Phe Cys Ala Phe Lys Ala Asp Gly Gly Arg CysArg Gly Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu GluPhe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu GluCys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:12) Met His Ser Phe Cys AlaPhe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ IDNO:13) Met His Ser Phe Cys Ala Phe Lys Ala Asp Val Gly Arg Cys Arg GlyAla His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe SerTyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp, (SEQ ID NO:14) Met His Ser Phe Cys Ala Phe LysAla Asp Val Gly Arg Cys Arg Gly Ala Gln Pro Arg Phe Phe Phe Asn Ile PheThr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn ArgPhe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:15)Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Ser Cys Arg Ala Ala HisLeu Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr GlyGly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys MetCys Thr Arg Asp, (SEQ ID NO:16) Met His Ser Phe Cys Ala Phe Lys Ala GluGly Gly Ser Cys Arg Ala Ala His Gln Arg Trp Phe Phe Asn Ile Phe Thr ArgGln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe GluSer Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:17) Met HisSer Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Gly Ala His Leu ArgPhe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly CysGly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys ThrArg Asp, (SEQ ID NO:18) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp GlyHis Cys Arg Gly Ala Leu Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser LeuGlu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:19) Met His Ser PheCys Ala Phe Lys Ala Asp Ser Gly Asn Cys Arg Gly Asn Leu Pro Arg Phe PhePhe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly GlyAsn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp,(SEQ ID NO:20) Met His Ser Phe Cys Ala Phe Lys Ala Asp Ser Gly Arg CysArg Gly Asn His Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu GluPhe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu GluCys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:21) Met His Ser Phe Cys AlaPhe Lys Ala Asp Gly Gly Arg Cys Arg Ala Ile Gln Pro Arg Trp Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ IDNO:22) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Arg Cys Arg GlyAla His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe SerTyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp.

Additional examples of peptides according to the present Invention arethose that differ (e.g., substitutions, insertions, or deletions) by atleast one amino acid, but fewer than seven, six, five, four, three, ortwo amino acids differences relative to an amino acid sequence describedherein, e.g., an amino acid sequence provided above. In one embodiment,fewer than three, two, or one differences are in one of the bindingloops. For example, the first binding loop may have no differencesrelative to an amino acid sequence described herein, e.g., an amino acidsequence provided above. In another example, neither the first nor thesecond binding loop differs from an amino acid sequence describedherein, e.g., an amino acid sequence provided above.

The peptide of the present invention can include (or consist of) apolypeptide described in U.S. Pat. No. 5,786,328, U.S. Pat. No.6,333,402 or U.S. Pat. No. 6,010,880, the content of which isincorporated by reference.

Examples of peptides according to the present Invention are described bythe following (where not indicated, “Xaa” refers to any amino acid, anynon-cysteine amino acid or any amino acid from the same set of aminoacids that are allowed for SEQ ID NO:1): (SEQ ID NO:23) Glu Ala Met HisSer Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro ArgTrp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly CysGlu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys ThrArg Asp, (SEQ ID NO:24) Glu Ala Met His Ser Phe Cys Ala Phe Lys AlaXaa10 Xaa11 Gly Xaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Arg Xaa21 PhePhe Asn Ile Phe Thr Arg Gln Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly Gly CysXaa39 Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met CysThr Arg Asp, (SEQ ID NO:25) Glu Ala Met His Ser Phe Cys Ala Phe Lys AlaAsp Asp Gly Pro Cys Lys Ala Asn His Leu Arg Phe Phe Phe Asn Ile Phe ThrArg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg PheGlu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:26) GluAla Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala AsnHis Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Thr TyrGly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys LysMet Cys Thr Arg Asp, (SEQ ID NO:27) Glu Ala Met His Ser Phe Cys Ala PheLys Ala Asp Asp Gly His Cys Lys Ala Asn His Gln Arg Phe Phe Phe Asn IlePhe Thr Arg Gln Cys Glu Gln Phe Thr Tyr Gly Gly Cys Ala Gly Asn Gln AsnArg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ IDNO:28) Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His CysLys Ala Ser Leu Pro Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu GluPhe Ile Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu GluCys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:29) Glu Ala Met His Ser PheCys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala Asn His Gln Arg Phe PhePhe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly GlyAsn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp,(SEQ ID NO:30) Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp GlyHis Cys Lys Gly Ala His Leu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser LeuGlu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:31) Glu Ala Met HisSer Phe Cys Ala Phe Lys Ala Asp Asp Gly Arg Cys Lys Gly Ala His Leu ArgPhe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly CysGlu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys ThrArg Asp, (SEQ ID NO:32) Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala AspGly Gly Arg Cys Arg Gly Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr ArgGln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe GluSer Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:33) Glu AlaMet His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala HisPro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr GlyGly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys MetCys Thr Arg Asp, (SEQ ID NO:34) Glu Ala Met His Ser Phe Cys Ala Phe LysAla Asp Val Gly Arg Cys Arg Gly Ala His Pro Arg Trp Phe Phe Asn Ile PheThr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn ArgPhe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:35)Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Val Gly Arg Cys Arg GlyAla Gln Pro Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe SerTyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp, (SEQ ID NO:36) Glu Ala Met His Ser Phe Cys AlaPhe Lys Ala Asp Asp Gly Ser Cys Arg Ala Ala His Leu Arg Trp Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ IDNO:37) Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Glu Gly Gly Ser CysArg Ala Ala His Gln Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu GluPhe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu GluCys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:38) Glu Ala Met His Ser PheCys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Gly Ala His Leu Arg Phe PhePhe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly GlyAsn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp,(SEQ ID NO:39) Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp GlyHis Cys Arg Gly Ala Leu Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser LeuGlu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:40) Glu Ala Met HisSer Phe Cys Ala Phe Lys Ala Asp Ser Gly Asn Cys Arg Gly Asn Leu Pro ArgPhe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly CysGly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys ThrArg Asp, (SEQ ID NO:41) Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala AspSer Gly Arg Cys Arg Gly Asn His Gln Arg Phe Phe Phe Asn Ile Phe Thr ArgGln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe GluSer Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:42) Glu AlaMet His Ser Phe Cys Ala Phe Lys Ala Asp Gly Gly Arg Cys Arg Ala Ile GlnPro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr GlyGly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys MetCys Thr Arg Asp, (SEQ ID NO:43) Glu Ala Met His Ser Phe Cys Ala Phe LysAla Asp Asp Gly Arg Cys Arg Gly Ala His Pro Arg Trp Phe Phe Asn Ile PheThr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn ArgPhe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp.

According to another embodiment, said peptide of the Invention thatinhibits serine protease includes (or consists of) the amino acidsequence:

Xaa-1 Xaa0 Xaa1 Xaa2 Xaa3 Xaa4 Cys Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 GlyXaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly GlyCys Xaa39 Xaa40 Xaa41 Xaa42 Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48 Xaa49Xaa50 Cys Xaa52 Xaa53 Xaa54 Cys Xaa56 Xaa57 Xaa58 (SEQ ID NO:44), or afragment or variant thereof, e.g. a fragment that binds and inhibitskallikrein wherein Xaa1 to Xaa58 are as defined above and Xaa-1 is Gluand Xaa0 is Ala.

According to preferred embodiment, the peptide of the Invention is SEQID NO:23 (Markland et al., 1996, Biochemistry, 35, 8058-8067 Ley et al.,1996, Mol Divers, 2, 119-124; U.S. Pat. No. 6,333,402).

The present invention also extends to the use of variants of the abovedisclosed peptides, said variants being more specifically defined assubstantially homologous to the peptides above disclosed. The term“substantially homologous”, when used in connection with amino acidsequences, refers to sequences which are substantially identical to orsimilar in sequence, giving rise to a homology in conformation and thusto similar biological activity. The term is not intended to imply acommon evolution of the sequences. Typically, “substantially homologous”sequences are at least 50% more preferably at least 80% identical insequence, at least over any regions known to be involved in the desiredactivity. Most preferably, no more than five residues, other than at thetermini, are different. Preferably, the divergence in sequence, at leastin the aforementioned regions, is in the form of “conservativemodifications”. “Conservative modifications” are defined as (i)conservative substitutions of amino acids as hereafter defined; and (ii)single or multiple insertions or deletions of amino acids at thetermini, at interdomain boundaries, in loops or in other segments ofrelatively high mobility (as indicated, e.g., by the failure to clearlyresolve their structure upon X-ray diffraction analysis or NMR).Preferably, except at the termini, no more than about five amino acidsare inserted or deleted at a particular locus, and the modifications areoutside regions known to contain binding sites important to activity.Conservative substitutions are herein defined as exchanges within one ofthe following five groups:

-   -   I. Small aliphatic, nonpolar or slightly polar residues: Ala,        Ser, Thr (Pro, Gly)    -   II. Polar, negatively charged residues: and their amides Asp,        Asn, Glu, Gln    -   III. Polar, positively charged residues: His, Arg, Lys    -   IV. Large, aliphatic, nonpolar residues: Met, Leu, Ile, Val        (Cys)    -   V. Large, aromatic residues: Phe, Tyr, Trp.

Residues Pro, Gly and Cys are parenthesized because they have specialconformational roles. Cys participates in formation of disulfide bonds.Gly imparts flexibility to the chain. Pro imparts rigidity to the chainand disrupts alpha helices. These residues may be essential in certainregions of the polypeptide, but substitutable elsewhere.

Semi-conservative substitutions are defined to be exchanges between twoof groups (I)-(V) above which are limited to supergroup (a), comprising(I), (II) and (III) above, or to supergroup (B), comprising (IV) and (V)above.

The compounds are not limited to the side groups found in geneticallyencoded amino acids; rather, conservative substitutions are allowed. Lyscan be replaced by Arg, ornithine, guamidolysine, and other side groupsthat carry a positive charge. Asn can be replaced by other small,neutral, hydrophilic groups, such as (but without limitation) Ser,O-methyl serine, Gln, alpha-amidoglycine, Ala, alpha-aminobutyric acid,and alpha-amino-gamma-hydroxybutyric acid (homoserine). His could bereplaced with other amino acids having one or more of the properties:amphoteric, aromatic, hydrophobic, and cyclic. For example (withoutlimitation), His could be replaced with methylhistidine,L-p-aminophenylalanine, L-m-(N,N,dimethylamino)phenylalanine, canavanineand N-methylasparagine.

The Kunitz domains are quite small; if this should cause apharmacological problem, such as excessively quick elimination from thecirculation, two or more such domains may be joined by a linker. Thislinker is preferably a sequence of one or more amino acids. Peptidelinkers have the advantage that the entire protein may then be expressedby recombinant DNA techniques. It is also possible to use a non-peptidyllinker, such as one of those commonly used to form immunogenicconjugates.

Chemical polypeptide synthesis is a well-described and practiced in theart. In general, as is known in the art, such methods involve blockingor protecting reactive functional groups, such as free amino, carboxyland thio groups. After polypeptide bond formation, the protective groupsare removed (or de-protected). Thus, the addition of each amino acidresidue requires several reaction steps for protecting and deprotecting.Current methods utilize solid phase synthesis, wherein the C-terminalamino acid is covalently linked to an insoluble resin particle largeenough to be separated from the fluid phase by filtration. Thus,reactants are removed by washing the resin particles with appropriatesolvents using an automated programmed machine. The completedpolypeptide chain is cleaved from the resin by a reaction which does notaffect polypeptide bonds.

The term “and/or” wherever used in the present Invention includes themeaning of “and”, “or” and “all or any other combination of the elementsconnected by said term”.

The terms “amino acids” and “residues” are synonyms and encompassnatural amino acids as well as amino acid analogs (e.g. non-natural,synthetic and modified amino acids, including D or L optical isomers).

The terms “polypeptide”, “peptide” and “protein” are used hereininterchangeably to refer to polymers of amino acid residues whichcomprise ten or more amino acids bonded via peptide bonds. The polymercan be linear, branched or cyclic and may comprise naturally occurringand/or amino acid analogs and it may be interrupted by non-amino acids.As a general indication, if the amino acid polymer is long (e.g. morethan 50 amino acid residues), it is preferably referred to as apolypeptide or a protein.

As used herein, the term “treatment” or “treating” encompassesprophylaxis and/or therapy. Accordingly the compositions and methods ofthe present invention are not limited to therapeutic applications andcan be used in prophylaxis ones. Therefore “treating” or “treatment” ofa state, disorder or condition includes: (i) preventing or delaying theappearance of clinical symptoms of the state, disorder or conditiondeveloping in a subject that may be afflicted with or predisposed to thestate, disorder or condition but does not yet experience or displayclinical or subclinical symptoms of the state, disorder or condition,(ii) inhibiting the state, disorder or condition, i.e., arresting orreducing the development of the disease or at least one clinical orsubclinical symptom thereof, or (iii) relieving the disease, i.e.causing regression of the state, disorder or condition or at least oneof its clinical or subclinical symptoms.

According to a specific embodiment, the peptides of the presentinvention are PEGylated, i.e. a plurality of polyethylene glycolmoieties are attached to the said peptide, especially those peptidesthat present available lysines and an N-terminus for modification withmPEG (see US20050089515).

According to a specific embodiment, the ophthalmic disorders of thepresent invention are exudative and/or inflammatory ophthalmicdisorders.

According to a specific embodiment, the ophthalmic disorders of thepresent invention are disorders related to impaired retinal vesselpermeability and/or integrity.

According to another specific embodiment, the ophthalmic disorders ofthe present invention are disorders related to retinal microvesselrupture leading to focal hemorrhages.

According to another embodiment, the ophthalmic disorders of the presentinvention are back of the eye diseases, and more specifically retinaldiseases.

According to another embodiment, the ophthalmic disorders of the presentinvention are front of the eye diseases.

According to the present Invention the terms “disease” and “disorder”have the same meaning.

Among the ophthalmic disorders (including exudative and/or inflammatoryophthalmic disorders, disorders related to impaired retinal vesselpermeability and/or integrity, disorders related to retinal microvesselrupture leading to focal hemorrhages, back of the eye diseases, retinaldiseases, and front of the eye diseases) which can be treated oraddressed in accordance with the present invention include, withoutlimitation, the following: Age Related Macular Degeneration (ARMD),exudative macular degeneration (also known as “wet” or neovascularage-related macular degeneration (wet-AMD), macular oedema, ageddisciform macular degeneration, cystoid macular oedema, palpebraloedema, retinal oedema, diabetic retinopathy, Acute MacularNeuroretinopathy, Central Serous Chorioretinopathy, chorioretinopathy,Choroidal Neovascularization, neovascular maculopathy, neovascularglaucoma, obstructive arterial and venous retinopathies (e.g. RetinalVenous Occlusion or Retinal Arterial Occlusion), Central Retinal VeinOcclusion, Disseminated Intravascular Coagulopathy, Branch Retinal VeinOcclusion, Hypertensive Fundus Changes, Ocular Ischemic Syndrome,Retinal Arterial Microaneurysms, Coat's Disease, ParafovealTelangiectasis, Hemi-Retinal Vein Occlusion, Papillophlebitis, CentralRetinal Artery Occlusion, Branch Retinal Artery Occlusion, CarotidArtery Disease(CAD), Frosted Branch Angitis, Sickle Cell Retinopathy andother Hemoglobinopathies, Angioid Streaks, macular oedema occurring as aresult of aetiologies such as disease (e.g. Diabetic Macular Oedema),eye injury or eye surgery; retinal ischemia or degeneration produced forexample by injury, trauma or tumours, uveitis, iritis, retinalvasculitis, endophthalmitis, panophthalmitis, metastatic ophthalmia,choroiditis, retinal pigment epithelitis, conjunctivitis, cyclitis,scleritis, episcleritis, optic neuritis, retrobulbar optic neuritis,keratitis, blepharitis, exudative retinal detachment, corneal ulcer,conjunctival ulcer, chronic nummular keratitis, Thygeson keratitis,progressive Mooren's ulcer, an ocular inflammatory disease caused bybacterial or viral infection, and by an ophthalmic operation, an ocularinflammatory disease caused by a physical injury to the eye, a symptomcaused by an ocular inflammatory disease including itching, flare,oedema and ulcer, erythema, erythema exsudativum multiforme, erythemanodosum, erythema annulare, scleroedema, dermatitis, angioneuroticoedema, laryngeal oedema, glottic oedema, subglottic laryngitis,bronchitis, rhinitis, pharyngitis, sinusitis, laryngitis or otitismedia.

According to the present invention, the term “back-of-eye diseases”refers to diseases affecting among other the retina, macular, fovea inthe posterior region of the eye. Examples of back-of-eye disease includemacular oedema such as clinical macular oedema or angiographic cystoidmacular oedema arising from various aetiologies such as diabetes,exudative macular degeneration and macular oedema arising from lasertreatment of the retina, age-related macular degeneration, retinopathyof prematurity (also known as retrolental fibroplasia), retinal ischemiaand choroidal neovascularization, retinal diseases (diabeticretinopathy, diabetic retinal oedema, retinal detachment, senile maculardegeneration due to sub-retinal neovascularization, myopic retinopathy);inflammatory diseases; uveitis associated with neoplasms such asretinoblastoma or pseudoglioma; neovascularization following vitrectomy;vascular diseases (retinal ischemia, choroidal vascular insufficiency,choroidal thrombosis, retinopathies resulting from carotid arteryischemia); neovascularization of the optic nerve.

According to the present invention, the term “front-of-eye” diseasesrefers to diseases affecting predominantly the tissues at thefront-of-eye, such as the cornea, iris, ciliary body, conjunctiva etc.Examples of front-of-eye diseases include corneal neovascularization(due to inflammation, transplantation, developmental hypoplasia of theiris, corneal diseases or opacifications with an exudative orinflammatory component, neovascularization due to penetration of the eyeor contusive ocular injury; chronic uveitis; anterior uveitis;inflammatory conditions resulting from surgeries such as LASIK, LASEK,refractive surgery, IOL implantation; irreversible corneal oedema as acomplication of cataract surgery; oedema as a result of insult or trauma(physical, chemical, pharmacological, etc); inflammation; conjunctivitis(eg. persistent allergic, giant papillary, seasonal intermittentallergic, perennial allergic, toxic, conjunctivitis caused by infectionby bacteria, viruses or Chlamydia); keratoconjunctivitis (vernal,atopic, Sicca); iridocyclitis; iritis; scleritis; episcleritis;infectious keratitis; superficial punctuate keratitis; keratoconus;posterior polymorphous dystrophy; Fuch's dystrophies (corneal andendothelial); aphakic and pseudophakic bullous keratopathy; cornealoedema; scleral disease; ocular cicatrcial pemphigoid; pars planitis;Posner Schlossman syndrome; Behcet's disease; Vogt-Koyanagi-Haradasyndrome; hypersensitivity reactions; ocular surface disorders;conjunctival oedema; Toxoplasmosis chorioretinitis; inflammatorypseudotumor of the orbit; chemosis; conjunctival venous congestion;periorbital cellulitis; acute dacryocystitis; non-specific vasculitis;sarcoidosis; cytomegalovirus infection.

In preferred embodiment, the Invention concerns back of the eyediseases.

According to the present invention, the term “therapeutically effectiveamount” is used herein to refer to an amount of therapeutic agent eitheras an individual compound or in combination with other compounds that issufficient to induce a therapeutic effect on the ailment which thecompound is applied to. This phrase should not be understood to meanthat the dose must completely eradicate the ailment. What constitutes atherapeutically effective amount will vary depending on, inter alia, thebiopharmacological properties of the compound used in the methodology,the condition being treated, the frequency of administration, the modeof delivery, characteristics of the individual to be treated theseverity of the disease and the response of the patient. These are thetypes of factors that a skilled pharmaceutical chemist will be aware ofand will be able to account for when formulating compositions for atreatment as described herein.

An effective quantity of the peptide of interest is preferably employedin the method of the invention. For ocular and extraocular formulations,the concentration of the peptide may be in the range of about 0.01% w/wto about 10% w/w. Typicaly, the concentration for this mode of deliveryis in the range of about 0.025% w/w to about 2.5% w/w.

The precise pharmaceutical formulation (i.e. ophthalmic composition)used in the method of the present invention will vary according to awide range of commercial and scientific criteria. That is the skilledreader will appreciate that the above formulation of the inventiondescribed above may contain other agents.

For example, the ophthalmic compositions used in the methods of theinvention are preferably prepared using a physiological saline solutionas a vehicle. The pH of the ophthalmic composition may be maintained ata substantially neutral pH (for example, about 7.4, in the range ofabout 6.5 to about 7.4, etc.) with an appropriate buffer system as knownto one skilled in the art (for example, acetate buffers, citratebuffers, phosphate buffers, borate buffers).

Any diluent used in the preparation of the ophthalmic composition maypreferably be selected so as not to unduly affect the biologicalactivity of the composition. Examples of such diluents which areespecially useful for injectable ophthalmic composition are water, thevarious saline, organic or inorganic salt solutions, Ringer's solution,dextrose solution, and Hank's solution.

In addition, the ophthalmic composition used in the method of theinvention may include additives such as other buffers, diluents,carriers, adjuvants or excipients. Any pharmacologically acceptablebuffer suitable for application to the eye may be used, e.g., tris orphosphate buffers. Other agents may be employed in the formulation for avariety of purposes. For example, buffering agents, preservatives,co-solvents, surfactants, oils, humectants, emollients, chelatingagents, stabilizers or antioxidants may be employed. Water solublepreservatives which may be employed include, but are not limited to,benzalkonium chloride, chlorobutanol, thimerosal, sodium bisulfate,phenylmercuric acetate, phenylmercuric nitrate, ethyl alcohol,methylparaben, polyvinyl alcohol, benzyl alcohol and phenylethylalcohol. A surfactant may be Tween 80.

Other vehicles that may be used include, but are not limited to,polyvinyl alcohol, povidone, hydroxypropyl methyl cellulose, poloxamers,carboxymethyl cellulose, hydroxyethyl cellulose, purified water, etc.Tonicity adjustors may be included, for example, sodium chloride,potassium chloride, mannitol, glycerin, etc. Antioxidants include, butare not limited to, sodium metabisulfite, sodium thiosulfate,acetylcysteine, butylated hydroxyanisole, butylated hydroxytoluene, etc.The indications, effective doses, formulations, contraindications,vendors etc. of the compounds in the ophthalmic composition areavailable or are known to one skilled in the art.

These agents may be present in individual amounts of from about 0.001%to about 5% by weight and preferably about 0.01% to about 2%. Suitablewater soluble buffering agents that may be employed are sodiumcarbonate, sodium borate, sodium phosphate, sodium acetate, sodiumbicarbonate, etc., as approved by the US FDA for the desired route ofadministration. These agents may be present in amounts sufficient tomaintain a pH of the system of between about 2 to about 9 and preferablyabout 4 to about 8. As such, the buffering agent may be as much as about5% (w/w) of the total ophthalmic composition. Electrolytes such as, butnot limited to, sodium chloride and potassium chloride may also beincluded in the formulation.

The ophthalmic composition of the present invention for the treatment orprevention of ophthalmic disorders may be provided in the form of asingle unit dose in a pre-prepared syringe, ready for administration.

In performing the method of the invention, ophthalmic composition may beadministered to a patient by any method that leads to delivery of thetherapeutic agent (i.e. the peptide of the Invention) to the site of theophthalmic condition (e.g. the location of an exudative retinopathy,inflammation or macular oedema). Any of the ophthalmic composition maybe administered by an ocular route, such as topical, subconjunctival,sub-Tenon, intraocular, ocular implants etc.

Administration of the ophthalmic composition to perform the method ofthe invention is preferably by intraocular injection, although othermodes of administration may be effective. Typically, ophthalmiccomposition will be delivered intraocularly (by chemical delivery systemor invasive device) to an individual. However, the invention is notlimited to intraocular delivery in that it also includes topically(extraocular application) or systemically (e.g. oral or other parenteralroute such as for example subcutaneous administration) provided that asufficient amount of the peptide within cells or tissue located in aneye or adjacent an eye achieves contact with the site of the ophthalmiccondition. Parenteral administration is used in appropriatecircumstances apparent to the practitioner. Preferably, the ophthalmiccompositions are administered in unit dosage forms suitable for singleadministration of precise dosage amounts.

As mentioned above, delivery to areas within the eye, in situ can beaccomplished by injection, cannula or other invasive device designed tointroduce precisely metered amounts of a desired ophthalmic compositionto a particular compartment or tissue within the eye (e.g. posteriorchamber or retina). An intraocular injection may be into the vitreous(intravitreal), or under the conjunctiva (subconjunctival), or behindthe eye (retrobulbar), into the sclera, or under the Capsule of Tenon(sub-Tenon), and may be in a depot form. Other intraocular routes ofadministration and injection sites and forms are also contemplated andare within the scope of the invention.

Preferably, the intraocular injection is an intravitreal injection,preferably through self sealing gauge needles or other any suitablycalibrated delivery device. Injection into the eye may be through thepars plana via the self-sealing needle.

In one embodiment, the ophthalmic composition is intraocularly injected(eg, into the vitreous) to treat or prevent an ophthalmic condition.When administering the ophthalmic composition by intravitreal injection,the active agents should be concentrated to minimise the volume forinjection. Preferably, the volume for injection is less than about 5 ml.Volumes such as this may require compensatory drainage of the vitreousfluid to prevent increases in intraocular pressure and leakage of theinjected fluid through the opening formed by the delivery needle. Morepreferably, the volume injected is between about 1.0 ml and 0.05 ml.Most preferably, the volume for injection is approximately 0.1 ml.

For injection, a concentration less than about 20 mg/ml may be injected,and any amount may be effective depending upon the factors previouslydescribed. Preferably a dose of less than 7 mg/ml is administered, withdoses of less than 6 mg/ml, 5 mg/ml, 4 mg/ml 3 mg/ml, 2 mg/ml and 1mg/ml being more preferred. Sample concentrations include, but are notlimited to, about 5 μg/ml to about 50 μg/ml; about 25 μg/ml to about 100μg/ml; about 100 μg/ml to about 200 μg/ml; about 200 μg/ml to about 500μg/ml; about 500 μg/ml to about 750 μg/ml; about 500 μg/ml up to 1 mg/mletc.

Intravitreal injection may be achieved by a variety of methods wellknown in the art. For example, the eye may be washed with a sterilisingagent such as Betadine® and the compound of the Invention is injected inan appropriate carrier with a fine gauge needle (e.g. 27 gauge) at aposition in the eye such that the compound will settle to the posteriorpole towards the ventral surface. It may be necessary to prepare the eyefor injection by application of positive pressure prior to injection. Insome cases, paracentesis may be necessary. Local anaesthetic or generalanaesthetic may be necessary.

The syringe used in practicing the method of this invention is suitablyone which can accommodate a 21 to 30 gauge needle (eg a 23, 24, 25, 26or 27 gauge needle) and is preferably of a small volume, for example 1.5ml, or more preferably 0.5 ml. Although it is possible that the needleand syringe may be of the type where the needle is removable from thesyringe, it is preferred that the arrangement is of a unitarysyringe/needle construction. This would clearly limit the possibility ofdisengagement of the needle from the syringe. It is also preferred thatthe arrangement be tamper evident. The formulations of the presentinvention may therefore be provided in the form of a single unit dose ina pre-prepared syringe, ready for administration.

A suitable style of syringe is, for example, sold under the name ofUniject® manufactured by Becton Dickinson and Company. In this style ofsyringe, the material is expelled through the needle into the eye bypressure applied to the sides of a pliable reservoir supplying theneedle, rather than by a plunger. As the name implies, the constructionof the reservoir and needle forms a single unit.

Topical application of ophthalmic composition of the invention for thetreatment or prevention of ophthalmic disorders may be as ointment, gelor eye drops. Preferably a penetrating composition comprising thepeptide(s) is used. The topical ophthalmic composition may further be anin situ gellable aqueous formulation. Such a formulation comprises agelling agent in a concentration effective to promote gelling uponcontact with the eye or with lacrimal fluid in the exterior of the eye.Suitable gelling agents include, but are not limited to, thermosettingpolymers such as tetra-substituted ethylene diamine block copolymers ofethylene oxide and propylene oxide (e.g., poloxamine); polycarbophil;and polysaccharides such as gellan, carrageenan (e.g., kappa-carrageenanand iota-carrageenan), chitosan and alginate gums.

The phrase “in situ gellable” as used herein embraces not only liquidsof low viscosity that form gels upon contact with the eye or withlacrimal fluid in the exterior of the eye, but also more viscous liquidssuch as semi-fluid and thixotropic gels that exhibit substantiallyincreased viscosity or gel stiffness upon administration to the eye.

To prepare a topical ophthalmic composition for the treatment ofophthalmic disorders, a therapeutically effective amount of theophthalmic composition of the invention is placed in an opthalmologicalvehicle as is known in the art. For example, topical ophthalmicformulations containing steroids are disclosed in U.S. Pat. No.5,041,434, whilst sustained release ophthalmic formulations of anophthalmic drug and a high molecular weight polymer to form a highlyviscous gel have been described in U.S. Pat. No. 4,271,143 and U.S. Pat.No. 4,407,792. Further GB 2007091 describes an ophthalmic composition inthe form of a gel comprising an aqueous solution of a carboxyvinylpolymer, a water-soluble basic substance and an ophthalmic drug.Alternatively, U.S. Pat. No. 4,615,697, discloses a controlled releasecomposition and method of use based on a bioadhesive and a treatingagent, such as an anti-inflammatory agent.

The amount of the peptide(s) to be administered and the concentration ofthe compound in the topical ophthalmic composition used in the methoddepend upon the diluent, delivery system or selected device, theclinical condition of the patient, the side effects and the stability ofthe compound in the formulation. Thus, the physician employs theappropriate preparation containing the appropriate concentration of thepeptide(s) and selects the amount of formulation administered, dependingupon clinical experience with the patient in question or with similarpatients.

Where the formulation contains two or more active agents (eg two or morepeptides, or a peptide and another agent such as a tetracyclinederivative etc), the active agents may be administered as a mixture, asan admixture, in the same ophthalmic composition, in separateformulations, in extended release formulations, liposomes,microcapsules, or any of the previously described embodiments. Theophthalmic composition may be administered topically, or may be injectedinto the eye, or one active agent may be administered topically and theother agent(s) may be injected.

The ophthalmic composition may be also administered as a slow releaseformulation, with a carrier formulation such as microspheres,microcapsules, liposomes, etc., as a topical ointment or solution, anintravenous solution or suspension, or in an intraocular injection, asknown to one skilled in the art to treat or prevent ophthalmicdisorders.

A time-release drug delivery system may be administered intraocularly toresult in sustained release of the agent over a period of time. Theophthalmic composition may be in the form of a vehicle, such as a micro-or macro-capsule or matrix of biocompatible polymers such aspolycaprolactone, polyglycolic acid, polylactic acid, polyanhydrides,polylactide-co-glycolides, polyamino acids, polyethylene oxide, acrylicterminated polyethylene oxide, polyamides, polyethylenes,polyacrylonitriles, polyphosphazenes, poly(ortho esters), sucroseacetate isobutyrate (SAIB), and other polymers such as those disclosedin U.S. Pat. Nos. 6,667,371; 6,613,355; 6,596,296; 6,413,536; 5,968,543;4,079, 038; 4,093,709; 4,131,648; 4,138,344; 4,180,646; 4,304,767;4,946,931, each of which is expressly incorporated by reference hereinin its entirety, or lipids that may be formulated as microspheres orliposomes. A microscopic or macroscopic ophthalmic composition may beadministered through a needle, or may be implanted by suturing withinthe eye, for example, within the lens capsule. Delayed or extendedrelease properties may be provided through various formulations of thevehicle (coated or uncoated microsphere, coated or uncoated capsule,lipid or polymer components, unilamellar or multilamellar structure, andcombinations of the above, etc.). The formulation and loading ofmicrospheres, microcapsules, liposomes, etc. and their ocularimplantation are standard techniques known by one skilled in the art,for example, the use a ganciclovir sustained-release implant to treatcytomegalovirus retinitis, disclosed in Vitreoretinal SurgicalTechniques, Peyman et al., Eds. (Martin Dunitz, London 2001, chapter45); Handbook of Pharmaceutical Controlled Release Technology, Wise, Ed.(Marcel Dekker, New York 2000), the relevant sections of which areincorporated by reference herein in their entirety. For example, asustained release intraocular implant may be inserted through the parsplana for implantation in the vitreous cavity.

The invention also provides a method for the treatment or prophylaxis ofophthalmic disorders with exudative/inflammatory conditions (e.g.exudative retinopathies), and/or ophthalmic disorders related toimpaired retinal vessel permeability and/or integrity, said methodcomprising the step of administering an ophthalmic compositioncomprising a therapeutically effective amount of at least one peptide ofthe Invention in a biocompatible, biodegradable matrix, for example inthe form of a gel or polymer which is preferably suited for insertioninto the retina or into a cavity of the eye, anterior or posterior, asan implant. In the case that the composition is delivered as an implant,it may be incorporated in any known biocompatible biodegradable matrixas a liquid, or in the form, for example, of a micelle using knownchemistry or as microparticles.

Slow or extended-release delivery systems include any of a number ofbiopolymers (biological-based systems), systems employing liposomes,colloids, resins, and other polymeric delivery systems orcompartmentalized reservoirs, can be utilized with the compositionsdescribed herein to provide a continuous or long term source oftherapeutic compound.

In any slow release device prepared, the said peptide(s) is preferablypresent in an amount of about 10% to 90% by weight of the implant. Morepreferably, the peptide(s) is from about 50% to about 80% by weight ofthe implant. In a preferred embodiment, the peptide(s) comprises about50% by weight of the implant. In a particularly preferred embodiment,the peptide(s) comprises about 70% by weight of the implant.

In one form, implants used in the method of the present invention areformulated with peptide(s) entrapped within the bio-erodible polymermatrix. Release of the agent is achieved by erosion of the polymerfollowed by exposure of previously entrapped agent particles to thevitreous, and subsequent dissolution and release of agent. The releasekinetics achieved by this form of drug release are different than thatachieved through formulations which release drug through polymerswelling, such as with hydrogels such as methylcellulose. In that case,the drug is not released through polymer erosion, but through polymerswelling, which releases drug as liquid diffuses through the pathwaysexposed. The parameters which determine the release kinetics include thesize of the drug particles, the water solubility of the drug, the ratioof drug to polymer, the method of manufacture, the surface area exposed,and the erosion rate of the polymer.

Exemplary biocompatible, non-biodegradable polymers of particularinterest include polycarbamates or polyureas, particularlypolyurethanes, polymers which may be cross-linked to producenon-biodegradable polymers such as cross-linked poly(vinyl acetate) andthe like. Also of particular interest are ethylene-vinyl estercopolymers having an ester content of 4% to 80% such as ethylene-vinylacetate (EVA) copolymer, ethylene-vinyl hexanoate copolymer,ethylene-vinyl propionate copolymer, ethylene-vinyl butyrate copolymer,ethylene-vinyl pentantoate copolymer, ethylene-vinyl trimethyl acetatecopolymer, ethylene-vinyl diethyl acetate copolymer, ethylene-vinyl3-methyl butanoate copolymer, ethylene-vinyl 3-3-dimethyl butanoatecopolymer, and ethylene-vinyl benzoate copolymer.

Additional exemplary naturally occurring or synthetic non-biodegradablepolymeric materials include poly(methylmethacrylate),poly(butylmethacrylate), plasticized poly(vinylchloride), plasticizedpoly(amides), plasticized nylon, plasticized soft nylon, plasticizedpoly(ethylene terephthalate), natural rubber, silicone, poly(isoprene),poly(isobutylene), poly(butadiene), poly(ethylene),poly(tetrafluoroethylene), poly(vinylidene chloride),poly(acrylonitrile, cross-linked poly(vinylpyrrolidone),poly(trifluorochloroethylene), chlorinated poly(ethylene),poly(4,4′-isopropylidene diphenylene carbonate), vinylidenechloride-acrylonitrile copolymer, vinyl chloridediethyl fumaratecopolymer, silicone, silicone rubbers (especially the medical grade),poly(dimethylsiloxanes), ethylene-propylene rubber, silicone-carbonatecopolymers, vinylidene chloride-vinyl chloride copolymer, vinylchloride-acrylonitrile copolymer, vinylidene chloride-acrylonitrilecopolymer, poly(olefins), poly(vinyl-olefins), poly(styrene),poly(halo-olefins), poly(vinyls), poly(acrylate), poly(methacrylate),poly(oxides), poly(esters), poly(amides), and poly(carbonates).

Diffusion of the peptide(s) from the implant may also be controlled bythe structure of the implant. For example, diffusion of the peptide(s)from the implant may be controlled by means of a membrane affixed to thepolymer layer comprising the drug. The membrane layer will be positionedintermediate to the polymer layer comprising the peptide(s) and thedesired site of therapy. The membrane may be composed of any of thebiocompatible materials indicated above, the presence of agents inaddition to the peptide(s) present in the polymer, the composition ofthe polymer comprising the peptide(s), the desired rate of diffusion andthe like. For example, the polymer layer will usually comprise a verylarge amount of peptide(s) and will typically be saturated. Suchpeptide(s)-saturated polymers may generally release the peptide(s) at avery high rate. In this situation, the release of the peptide(s) may beslowed by selecting a membrane which is of a lower peptide(s)permeability than the polymer. Due to the lower peptide(s) permeabilityof the membrane, the peptide(s) will remain concentrated in the polymerand the overall rate of diffusion will be determined by the peptide(s)permeability of the membrane. Therefore, the rate of release of thepeptide(s) from the implant is reduced, providing for a more controlledand extended delivery of the peptide(s) to the site of therapy.

The skilled reader will appreciate that the duration over which any ofthe ophthalmic compositions used in the method of the invention willdwell in the ocular environment will depend, inter alia, on such factorsas the physicochemical and/or pharmacological properties of thecompounds employed in the formulation, the concentration of the compoundemployed, the bioavailability of the compound, the disease to betreated, the mode of administration and the preferred longevity of thetreatment. Where that balance is struck will often depend on thelongevity of the effect required in the eye and the ailment beingtreated.

The frequency of treatment according to the method of the invention isdetermined according to the disease being treated, the deliverableconcentration of the peptide(s) and the method of delivery. Ifdelivering the peptide(s) by intravitreal injection, the dosagefrequency may be monthly. Preferably, the dosage frequency is everythree months. The frequency of dosage may also be determined byobservation, with the dosage being delivered when the previouslydelivered peptide(s) is visibly cleared. Once a therapeutic result isachieved, the peptide(s) can be tapered or discontinued. Occasionally,side effects warrant discontinuation of therapy. In general, aneffective amount of the compound is that which provides eithersubjective relief of symptoms or an objectively identifiable improvementas noted by the clinician or other qualified observer.

Ophthalmic compositions prepared for used in the method of the presentinvention to prevent or treat ophthalmic disorders will preferably havedwell times from hours to many months and possibly years, although thelatter time period requires special delivery systems to attain such aduration. Illustrative forms of such delivery systems are disclosedelsewhere in this specification (eg below). Most preferably theformulations for use in the method of the invention will have a dwelltime (ie duration in the eye) of hours (i.e. 1 to 24 hours), days (i.e.1, 2, 3, 4, 5, 6 or 7 days) or weeks (i.e. 1, 2, 3, 4 weeks).Alternatively, the formulation will have a dwell time of at least a fewmonths such as, 1 month, 2 months, 3 months, with dwell times of greaterthan 4, 5, 6, 7 to 12 months being achievable.

The methods of treatment or prophylaxis of ophthalmic conditions of thepresent invention may be performed alone, or in combination with one ormore other therapies such as photodynamic therapy, laser surgery, laserphotocoagulation or one or more biological or pharmaceutical treatments.

Laser treatment takes a number of forms, depending on the nature of theophthalmic disorder. Disorders such as myopia may be treated with lasersurgery to reshape the cornea (eg. LASIK® surgery), whilst a widely usedtreatment for disorders such as AMD is laser therapy which is directedto removal or blockage of blood vessels via photodynamic therapy orlaser photocoagulation. Laser therapy may further be used to treat orremove neoplasm such as retinoblastomas or pseudogliomas.

Photocoagulation involves the use of a laser to seal leaking bloodvessels, slow the growth of abnormal blood vessels and/or destroy newblood vessels within the eye. In addition, the laser can be used to sealthe retina to the eye, helping to prevent retinal detachment. Forexample, focal laser treatment may be applied to microaneurysmsidentified in diabetic retinopathy.

Photodynamic therapy involves the use of a photoactive drug (egVisudyne®) and a laser to destroy abnormal blood vessels. Visudyne® isinjected into the blood and activated with a laser, effectivelydestroying the blood vessels. This treatment may require severalsessions to be effective. A wide range of theories have been proposed toexplain the beneficial effects of retinal laser photocoagulation indelaying retinal angiogenesis, however, the underlying molecularmechanism remains to be elucidated.

The therapeutic effects of laser photocoagulation are thought to be dueto the destruction of photoreceptors, the highest oxygen consumers inthe retina. Subsequently, these photoreceptors are replaced by glialcells allowing increased oxygen diffusion from the choroid to the innerretina thereby relieving inner retinal hypoxia. This improvedoxygenation triggers a two-pronged cascade of events where: (1)constriction of the retinal arteries results in decreased hydrostaticpressure in capillaries and the constriction of capillaries and venules;and (2) the cellular production of VEGF is inhibited. Together, theseeffects are believed to ultimately result in the inhibition ofneovascularization and a decrease in oedema. Cell proliferation andregulation of cellular proteins are induced by the laserphotocoagulation, and their therapeutic effect might be an essentialpart of the physiological response.

However, a complication of laser treatment (either photodynamic lasertherapy or laser photocoagulation) is inflammation, leading to furtheroedema. This may also occur after laser therapy to remove or treatocular neoplasm. In addition, laser treatment is not always a permanentcure as the blood vessels may begin to grow again, and microaneurysmsmay reform. Furthermore, laser treatment of abnormal blood vesselscannot be performed on vessels located in certain regions of the retina,such as the central region of the macula.

Therefore, in an embodiment of the invention, where laser treatment ofthe retina is indicted, administration of an ophthalmic composition ofthe Invention may be carried out by injection before or after the lasertreatment. Administration of the peptide(s) of the Invention may reduce,eliminate or prevent oedema before or after laser therapy and maytherefore reduce or eliminate one of the side effects of laser therapy.

In another embodiment, the Invention resides in a method for reducingocular irritation comprising the step of administering to a patient anophthalmic composition of the Invention to a patient following cornealsurgery (e.g., LASIK® surgery, photorefractive keratectomy (PRK), orother corneal procedures). Such treatment reduces or inhibits theexudation of fluids in the eye which may cloud the cornea or thevitreous.

In addition to the other compounds previously described, ophthalmiccomposition of the invention may further comprise anti-angiogenic agentsdesigned to block the actions of VEGF on endothelial cells in combinedtherapies. Examples of agents that can be employed in the method of theinvention are: (a) Lucentis® developed by Genentech; and (b) Macugen®developed by Eyetech Pharmaceuticals. Lucentis® and Macugen® arecompounds that are injected into the vitreous and are potentanti-angiogenic compounds.

In another aspect of the invention, the ophthalmic composition of theinvention may further comprise a compound selected in the groupconsisting of a glucocorticoid (e.g. prednisolone, prednisone), anoestrogen (e.g. oestrodiol), an androgen (e.g. testosterone) retinoicacid derivatives (e.g. 9-cis-retinoic acid, 13-trans-retinoic acid,all-trans retinoic acid), a vitamin D derivative (e.g. calcipotriol,calcipotriene), a non-steroidal anti-inflammatory agent, a vitamin Dderivative, an anti-infective agent, a protein kinase C inhibitor, a MAPkinase inhibitor, an anti-apoptotic agent, a growth factor, a nutrientvitamin, an unsaturated fatty acid, and/or ocular anti-infective agents,for the treatment of the ophthalmic disorders set forth herein. In stillother embodiments of the invention, a mixture of these agents may beused. Ocular anti-infective agents that may be used include, but are notlimited to, penicillins (ampicillin, aziocillin, carbenicillin,dicloxacillin, methicillin, nafcillin, oxacillin, penicillin G,piperacillin, and ticarcillin), cephalosporins (cefamandole, cefazolin,cefotaxime, cefsulodin, ceftazidime, ceftriaxone, cephalothin, andmoxalactam), aminoglycosides (amikacin, gentamicin, netilmicin,tobramycin, and neomycin), miscellaneous agents such as aztreonam,bacitracin, ciprofloxacin, clindamycin, chloramphenicol, cotrimoxazole,fusidic acid, imipenem, metronidazole, teicoplanin, and vancomycin),antifungals (amphotericin B, clotrimazole, econazole, fluconazole,flucytosine, itraconazole, ketoconazole, miconazole, natamycin,oxiconazole, and terconazole), antivirals (acyclovir, ethyldeoxyuridine,foscarnet, ganciclovir, idoxuridine, trifluridine, vidarabine, and(S)-1-(3-dydroxy-2-phospho-nyluethoxypropyl)cytosine (HPMPC)),antineoplastic agents (cell cycle (phase) nonspecific agents such asalkylating agents (chlorambucil, cyclophosphamide, mechlorethamine,melphalan, and busulfan), anthracycline antibiotics (doxorubicin,daunomycin, and dactinomycin), cisplatin, and nitrosoureas),antimetabolites such as antipyrimidines (cytarabine, fluorouracil andazacytidine), antifolates (methotrexate), antipurines (mercaptopurineand thioguanine), bleomycin, vinca alkaloids (vincrisine andvinblastine), podophylotoxins (etoposide (VP-16)), and nitrosoureas(carmustine, (BCNU)), immunosuppressant agents such as cyclosporin A andSK506, and anti-inflammatory or suppressive agents (inhibitors), andinhibitors of proteolytic enzymes such as plasminogen activatorinhibitors. Doses for topical and sub-conjunctival administration of theabove agents, as well as intravitreal dose and vitreous half-life may befound in Intravitreal Surgery Principles and Practice, Peyman G A andShulman, J. Eds., 2nd edition, 1994, Appleton-Longe, the relevantsections of which are expressly incorporated by reference herein.

According to another embodiment, the invention provides methods of usingserine protease inhibitors in a method for treating and/or preventingophthalmic disorders and compositions for such use. According to anotherembodiment, the invention provides methods of using kallikreininhibitors in a method for treating and/or preventing ophthalmicdisorders and compositions for such use. Examples of said inhibitors arepeptides such as those disclosed above, or inhibitors selected amongdirect and indirect inhibitors. The term “direct inhibitor” as usedherein, refers to an agent able to interfere with the production ofbradykinin and/or kallidin. It relates to an agent able to decrease(e.g. by at least 10%, 20%, or 30% or more) the activity of kallikreineither in vitro or in vivo after administration to a mammal, such as ahuman. According to a more preferred embodiment, said direct inhibitoris an agent which decreases (e.g. by at least 10%, 20%, or 30%,preferably 50%, more preferably 75% or 85%, and most preferably 95%) thekininogenase activity of kallikrein. These functional characterisationsof the direct inhibitor can be tested using well known assay methods,such as for example those disclosed in Gallimore et al, 1979, Thromb Res16, 695-703; Kondo et al., 1984, Endocrinol Jpn., 31, 635-643. “Partialinhibitor” refers to a compound which acts as the inhibitor but thatproduces a weak maximum inhibitory response. This term is well known inthe art. Exemplary kallikrein inhibitors (e.g. plasma kallikreininhibitors) include those described in U.S. Pat. No. 6,333,402, U.S.Pat. No. 6,057,287, U.S. Pat. No. 6,010,880 or Zhang et al., 2006, Med.Chem., 2, 545-553 the contents of which are incorporated herein byreference in their entirety.

The term “indirect inhibitor” as used herein, refers for example to anagent able to interfere specifically with the kallikrein geneexpression, and more particularly with the kallikrein mRNA. According toone embodiment of the present invention, the said inhibitor or partialinhibitor is selected in the group consisting of antisense RNA, siRNA,ribozyme, miRNA, shRNA, i.e. compounds that reduce the expression levelsof said kallikrein, preferably plasma kallikrein. According to anotherembodiment, the term “indirect inhibitor” as used herein, refers to ananti-kallikrein or anti-prekallikrein antibody. The term “antibody” asused herein refers to an immunoglobulin molecule or immunologicallyactive portion thereof, i.e., an antigen-binding portion. Examples ofimmunologically active portions of immunoglobulin molecules include scFVand dcFV fragments, Fab and F (ab′) 2 fragments which can be generatedby treating the antibody with an enzyme such as papain or pepsin,respectively. The antibody can be a polyclonal, monoclonal, recombinant,e.g. a chimeric or humanized, fully human, non-human, e.g. murine orsingle chain antibody. The antibody can be coupled to a toxin or imagingagent. Additionally, chimeric, humanized, and completely humanantibodies are also within the scope of the invention. Chimeric,humanized, but most preferably, completely human antibodies aredesirable for applications which include repeated administration, e.g.,therapeutic treatment of human patients. These terms and methods forproducing these antibodies by recombinant DNA techniques are widelyknown in the art (see for example EP184187, EP171496, EP173494, WO86/01533, U.S. Pat. No. 4,816,567).

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. The invention includes all such variation andmodifications. The invention also includes all of the steps, features,formulations and compounds referred to or indicated in thespecification, individually or collectively and any and all combinationsor any two or more of the steps or features.

Each document, reference, patent application or patent cited in thistext is expressly incorporated herein in their entirety by reference,which means that it should be read and considered by the reader as partof this text. That the document, reference, patent application or patentcited in this text is not repeated in this text is merely for reasons ofconciseness.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification only. Functionally equivalent products, formulations andmethods are clearly within the scope of the invention as describedherein.

The invention described herein may include one or more range of values(eg size, concentration etc). A range of values will be understood toinclude all values within the range, including the values defining therange, and values adjacent to the range which lead to the same orsubstantially the same outcome as the values immediately adjacent tothat value which defines the boundary to the range.

EXAMPLES Figures

FIG. 1—Effect of intra-vitreous SEQ. ID. N^(o)23 on OCT-measured maximalretinal thickness in a pig model of RVO.

Maximal retina thickness was determined 24 h after RVO from OpticalCoherence Tomography images. Values are mean ±s.e.mean. Comparison ofvalues was performed by a one-way ANOVA following by a student t-test.

FIG. 2—Effect of intra-vitreous SEQ. ID. N^(o)23 on the development ofextra-cellular retinal oedema in a pig model of RVO.

The amount of Evans Blue dye concentration into the retinal tissuereflects plasma extravasation and the extent of oedema. Control valuesrepresent retina Evans Blue Dye content of 2 eyes that were leftnon-operated and non-treated whilst sham-operated eyes (n=4) weresubmitted to the surgical procedure but without occlusion (See Methods).Values are mean ±s.e.mean. Comparison of values was performed by aone-way ANOVA following by a student t-test. **, p<0.01

Example 1 Materials and Methods

Pig Preparation

Mixed breed (Large White×Landrace×Pietrain) female pigs weighing 50 to60 kg aged of 5 to 6 months were used. Following intramuscular injectionof ketamine (10 mg/kg), azaperone (2 mg/kg) and atropine (0.02 mg/kg),anesthesia was induced by intravenous sodium thiopental (10 mg/kg).After tracheal intubation, anesthesia was maintained with isoflurane(1-2% in 100% O₂) using a Hallowell ventilator (15 to 20 rpm; pressureat 20 cmH₂O)-Three-lead ECG (lead II configuration), body temperature,arterial blood pressure and blood gases were continuously monitored.

Procedure of Retinal Vein Occlusion (RVO)

Pupil was dilated by tropicamide. Cunjunctival disinsertion was followedby a 0.9 mm sclerotomy, 3 mm from the limbus. The fundus was observedusing a plano-concave lens and the axial light of the operatingmicroscope (Microscope OPMI 6-C, Zeiss, Germany). Branch retinal veinocclusion (RVO) of the major temporal vein was performed by transvitrealcauterization using a 300 micron probe (GN 300, Aesculap, Tuttlingen,Germany). Completion of the occlusion was assessed by the completearrest of blood flow upstream of the occlusion site. Both eyes weresubmitted to RVO.

Sham-operation was performed in two pigs (4 eyes) as follows pupil wasdilated by tropicamide. Cunjunctival disinsertion was followed by a 0.9mm sclerotomy, 3 mm from the limbus. The fundus was observed using aplano-concave lens and the axial light of the operating microscope(Microscope OPMI 6-C, Zeiss, Germany).

One pig was non-operated and non-treated.

Drug Treatments

The number of animals tested has been increased.

Animals that were submitted to RVO were randomised to receive either thevehicle or the drug (10 to 14 pigs/group). Accordingly, 100 μl of SEQ IDN^(o)23 in solution (dosage of 21.2 μg/eye) or of the correspondingvehicle (saline) was injected intra-vitreally, immediately followingRVO.

SEQ ID N^(o)23 (also named DX 88 in Figures) is a peptide of thefollowing sequence: Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp AspGly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg GlnCys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu SerLeu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp

Measurement of Retinal Maximal Thickness and Oedema

Twenty four hours later, the animals will be anesthetized again, and a40 MHz ultrasonographic examination of the posterior retina will beperformed. The scan will be oriented so as to encompass the normal andedematous retina. This will ensure an optimal placement of the OCT scan.Optical coherence tomography (Stratus OCT, Zeiss Humphrey, Dublin,Calif.) will document the maximal thickening of the central retinaand/or the presence of subretinal fluid.

After OCT measurement, each animal received Evans Blue dye at 45 mg/kgi.v. and venous blood samples (approximately 1 ml) were obtained at 15minutes intervals for 2 hours. These blood samples were centrifuged at12,000 rpm for 15 min. After the dye had circulated for 2 hours, theanimals were infused for 10 minutes via the left cardiac ventricle withcitrate buffer (0.05 M, pH 3.5) and blood was collected from the rightcardiac ventricle. The whole infusion volume was 51 over 10 minutes.After infusion, both eyes were enucleated and bisected at equator. Theretinas were carefully dissected out. Retina were prepared, weighed anddesiccated in Speed-Vac for 5 hours. Evans Blue dye was extracted byincubating each retina in 500 μl formamide for 18 hours at 70° C. Thesupernatant was filtered through Ultrafree-MC at 3,000 rpm for 2 hours.Evans Blue dye concentration in plasma samples and in retina tissue wasmeasured by spectrophotometry at both 620 nm and 740 nm.

Thus, in an embodiment of the present invention, a model of RVO in thepig is described which allows evaluation of drugs on retinal thicknessand oedema. An example is given with the evaluation of SEQ ID N^(o)23 inthis model.

Example 2 Effect of SEQ. ID. N^(o)23 in a Pig Model of Acute MacularOedema Induced by RVO

At 24 h, spontaneous reperfusion of retinal vein was observed in 10% and30% of vehicle—and SEQ ID N^(o)23-treated eyes, respectively (See Table1 below).

Evans Blue dye retinal concentration, which represents the extent ofextra-cellular oedema, was markedly increased 24 h following RVO (FIG.2). This was significantly (p<0.01) reduced by 47% in SEQ. ID.N^(o)23-treated pigs (FIG. 1).

After repeated experiments, we concluded that in the animal modelselected, the peptide SEQ ID N^(o)23 did not modify the increase ofmaximal retinal thickness as measured by OCT (FIG. 1). We concluded thatthe method of measurement used was actually not adapted to the presentanimal model. Our interpretation is that SEQ. ID. N^(o)23 specificallytargets vascular leakage which appears to be minor in respect to theischemic oedema component in the pig RVO model. In addition, OCT imagesrevealed that the retina presents an irregular thickening and bumpysurface after RVO occlusion that makes OCT-based measurements poorlyreliable. TABLE 1 Effect of SEQ. ID. N^(o) 23 on the rate of spontaneousretinal vein reperfusion 24 h after RVO in the pig Number of pigs/eyesNumber (%) of reperfused submitted to RVO retinal vein at 24 h Vehicle10/20 2 (10%) SEQ. ID. N^(o) 23 13/26 8 (30%)

1: A method inhibits serine protease for the preparation of anophthalmic composition useful for the treatment of ophthalmic disordersin a patient in need thereof comprising utilizing at least one peptidethat inhibits serine protease. 2: The method of claim 1, wherein saidpeptide is a kallikrein inhibitor. 3: The method of claim 1, whereinsaid peptide is a Kunitz domain polypeptide. 4: The method of claim 1,wherein said peptide includes the amino acid sequence: Xaa1 Xaa2 Xaa3Xaa4 Cys Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Gly Xaa13 Cys Xaa15 Xaa16 Xaa17Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly Gly Cys Xaa39 Xaa40 Xaa41 Xaa42Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48 Xaa49 Xaa50 Cys Xaa52 Xaa53 Xaa54Cys Xaa56 Xaa57 Xaa58 (SEQ ID NO:1) with Xaa_(s) being, independentlyfrom one another, any amino acid. 5: The method of claim 4, wherein oneor more of the following apply: Xaa1, Xaa2, Xaa3, Xaa4, Xaa56, Xaa57 orXaa58 are, independently from one another, any amino acid or absent;Xaa10 is an amino acid selected from the group consisting of Asp andGlu; Xaa11 is an amino acid selected from the group consisting of Asp,Gly, Ser, Val, Asn, lie, Ala and Thr; Xaa13 is an amino acid selectedfrom the group consisting of Arg, His, Pro, Asn, Ser, Thr, Ala, Gly, Lysand Gln; Xaa15 is an amino acid selected from the group consisting ofArg, Lys, Ala, Ser, Gly, Met, Asn and Gln; Xaa16 is an amino acidselected from the group consisting of Ala, Gly, Ser, Asp and Asn; Xaa17is an amino acid selected from the group consisting of Ala, Asn, Ser,Ile, Gly, Val, Gln and Thr; Xaa18 is an amino acid selected from thegroup consisting of His, Leu, Gln and Ala; Xaa19 is an amino acidselected from the group consisting of Pro, Gln, Leu, Asn and Ile; Xaa21is an amino acid selected from the group consisting of Trp, Phe, Tyr,His and Ile; Xaa22 is an amino acid selected from the group consistingof Tyr and Phe; Xaa23 is an amino acid selected from the groupconsisting of Tyr and Phe; Xaa31 is an amino acid selected from thegroup consisting of Glu, Asp, Gln, Asn, Ser, Ala, Val, Leu, Ile and Thr;Xaa32 is an amino acid selected from the group consisting of Glu, Gln,Asp Asn, Pro, Thr, Leu, Ser, Ala, Gly and Val; Xaa34 is an amino acidselected from the group consisting of Thr, Ile, Ser, Val, Ala, Asn, Glyand Leu; Xaa35 is an amino acid selected from the group consisting ofTyr, Trp and Phe; Xaa39 is an amino acid selected from the groupconsisting of Glu, Gly, Ala, Ser and Asp; Xaa40 is an amino acidselected from the group consisting of Gly and Ala; Xaa43 is an aminoacid selected from the group consisting of Asn and Gly; Xaa45 is anamino acid selected from the group consisting of Phe and Tyr; Xaa6,Xaa7, Xaa8, Xaa9, Xaa20, Xaa24, Xaa25, Xaa26, Xaa27, Xaa28, Xaa29,Xaa41, Xaa42, Xaa44, Xaa46, Xaa47, Xaa48, Xaa49, Xaa50, Xaa52, Xaa53 andXaa54 are, independently from one another, any amino acid. 6: The methodof claim 1, wherein said peptide is selected in the group consisting of:(SEQ ID NO:2) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro CysArg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu GluPhe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu GluCys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:3) Met His Ser Phe Cys AlaPhe Lys Ala Xaa10 Xaa11 Gly Xaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 ArgXaa21 Phe Phe Asn Ile Phe Thr Arg Gln Cys Xaa31 Xaa32 Phe Xaa34 Xaa35Gly Gly Cys Xaa39 Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp, (SEQ ID NO:4) Met His Ser Phe Cys Ala Phe LysAla Asp Asp Gly Pro Cys Lys Ala Asn His Leu Arg Phe Phe Phe Asn Ile PheThr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn ArgPhe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:5)Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala Asn HisGln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Thr Tyr GlyGly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys MetCys Thr Arg Asp, (SEQ ID NO:6) Met His Ser Phe Cys Ala Phe Lys Ala AspAsp Gly His Cys Lys Ala Asn His Gln Arg Phe Phe Phe Asn Ile Phe Thr ArgGln Cys Glu Gln Phe Thr Tyr Gly Gly Cys Ala Gly Asn Gln Asn Arg Phe GluSer Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:7) Met HisSer Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala Ser Leu Pro ArgPhe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly CysGly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys ThrArg Asp, (SEQ ID NO:8) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp GlyHis Cys Lys Ala Asn His Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser LeuGlu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:9) Met His Ser PheCys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Gly Ala His Leu Arg Phe PhePhe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu GlyAsn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp,(SEQ ID NO:10) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Arg CysLys Gly Ala His Leu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu GluPhe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu GluCys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:11) Met His Ser Phe Cys AlaPhe Lys Ala Asp Gly Gly Arg Cys Arg Gly Ala His Pro Arg Trp Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ IDNO:12) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg AlaAla His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe SerTyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp, (SEQ ID NO:13) Met His Ser Phe Cys Ala Phe LysAla Asp Val Gly Arg Cys Arg Gly Ala His Pro Arg Trp Phe Phe Asn Ile PheThr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn ArgPhe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:14)Met His Ser Phe Cys Ala Phe Lys Ala Asp Val Gly Arg Cys Arg Gly Ala GlnPro Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr GlyGly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys MetCys Thr Arg Asp, (SEQ ID NO:15) Met His Ser Phe Cys Ala Phe Lys Ala AspAsp Gly Ser Cys Arg Ala Ala His Leu Arg Trp Phe Phe Asn Ile Phe Thr ArgGln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe GluSer Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:16) Met HisSer Phe Cys Ala Phe Lys Ala Glu Gly Gly Ser Cys Arg Ala Ala His Gln ArgTrp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly CysGly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys ThrArg Asp, (SEQ ID NO:17) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp GlyPro Cys Arg Gly Ala His Leu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser LeuGlu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:18) Met His Ser PheCys Ala Phe Lys Ala Asp Asp Gly His Cys Arg Gly Ala Leu Pro Arg Trp PhePhe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly GlyAsn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp,(SEQ ID NO:19) Met His Ser Phe Cys Ala Phe Lys Ala Asp Ser Gly Asn CysArg Gly Asn Leu Pro Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu GluPhe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu GluCys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:20) Met His Ser Phe Cys AlaPhe Lys Ala Asp Ser Gly Arg Cys Arg Gly Asn His Gln Arg Phe Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ IDNO:21) Met His Ser Phe Cys Ala Phe Lys Ala Asp Gly Gly Arg Cys Arg AlaIle Gln Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe SerTyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp, (SEQ ID NO:22) Met His Ser Phe Cys Ala Phe LysAla Asp Asp Gly Arg Cys Arg Gly Ala His Pro Arg Trp Phe Phe Asn Ile PheThr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn ArgPhe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp. (SEQ ID NO:23)Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg AlaAla His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe IleTyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp, (SEQ ID NO:24) Glu Ala Met His Ser Phe Cys AlaPhe Lys Ala Xaa10 Xaa11 Gly Xaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 ArgXaa21 Phe Phe Asn Ile Phe Thr Arg Gln Cys Xaa31 Xaa32 Phe Xaa34 Xaa35Gly Gly Cys Xaa39 Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp, (SEQ ID NO:25) Glu Ala Met His Ser Phe Cys AlaPhe Lys Ala Asp Asp Gly Pro Cys Lys Ala Asn His Leu Arg Phe Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ IDNO:26) Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His CysLys Ala Asn His Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu GluPhe Thr Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu GluCys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:27) Glu Ala Met His Ser PheCys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala Asn His Gln Arg Phe PhePhe Asn Ile Phe Thr Arg Gln Cys Glu Gln Phe Thr Tyr Gly Gly Cys Ala GlyAsn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp,(SEQ ID NO:28) Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp GlyHis Cys Lys Ala Ser Leu Pro Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu Phe Ile Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser LeuGlu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:29) Glu Ala Met HisSer Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala Asn His Gln ArgPhe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly CysGly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys ThrArg Asp, (SEQ ID NO:30) Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala AspAsp Gly His Cys Lys Gly Ala His Leu Arg Phe Phe Phe Asn Ile Phe Thr ArgGln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe GluSer Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:31) Glu AlaMet His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Arg Cys Lys Gly Ala HisLeu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr GlyGly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys MetCys Thr Arg Asp, (SEQ ID NO:32) Glu Ala Met His Ser Phe Cys Ala Phe LysAla Asp Gly Gly Arg Cys Arg Gly Ala His Pro Arg Trp Phe Phe Asn Ile PheThr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn ArgPhe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:33)Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg AlaAla His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe SerTyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp, (SEQ ID NO:34) Glu Ala Met His Ser Phe Cys AlaPhe Lys Ala Asp Val Gly Arg Cys Arg Gly Ala His Pro Arg Trp Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ IDNO:35) Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Val Gly Arg CysArg Gly Ala Gln Pro Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu GluPhe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu GluCys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:36) Glu Ala Met His Ser PheCys Ala Phe Lys Ala Asp Asp Gly Ser Cys Arg Ala Ala His Leu Arg Trp PhePhe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly GlyAsn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp,(SEQ ID NO:37) Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Glu Gly GlySer Cys Arg Ala Ala His Gln Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser LeuGlu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:38) Glu Ala Met HisSer Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Gly Ala His Leu ArgPhe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly CysGly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys ThrArg Asp, (SEQ ID NO:39) Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala AspAsp Gly His Cys Arg Gly Ala Leu Pro Arg Trp Phe Phe Asn Ile Phe Thr ArgGln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe GluSer Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:40) Glu AlaMet His Ser Phe Cys Ala Phe Lys Ala Asp Ser Gly Asn Cys Arg Gly Asn LeuPro Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr GlyGly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys MetCys Thr Arg Asp, (SEQ ID NO:41) Glu Ala Met His Ser Phe Cys Ala Phe LysAla Asp Ser Gly Arg Cys Arg Gly Asn His Gln Arg Phe Phe Phe Asn Ile PheThr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn ArgPhe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO:42)Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Gly Gly Arg Cys Arg AlaIle Gln Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe SerTyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp, (SEQ ID NO:43) Glu Ala Met His Ser Phe Cys AlaPhe Lys Ala Asp Asp Gly Arg Cys Arg Gly Ala His Pro Arg Trp Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp.

7: The method of claim 1, wherein said peptide is Glu Ala Met His SerPhe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg TrpPhe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys GluGly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr ArgAsp (SEQ ID NO:23). 8: The method of claim 1, wherein said ophthalmicdisorder is an exudative and/or inflammatory ophthalmic disorder. 9: Themethod of claim 1, wherein said ophthalmic disorder is related toimpaired retinal vessel permeability and/or integrity. 10: The method ofclaim 1, wherein said ophthalmic disorder is a back of the eye disease.